This work was supported by FIRB-Merit grants RBNE08YFN3_005 and RBNE08HWLZ_012, and the Italian Ministry of Economy and Finance to the CNR for the Project &#8220;FaReBio di Qualit&#224;&#8221;.

1.のsiRNA溶液を調製します<ol><li>再懸 ​​濁5スクランブルのナノモル（Ctrlキー）とRNaseフリー水（最終siRNA濃度：50μM）の100μl中BRCA2 siRNAを乾燥したペレットの5ナノモル。これは、siRNAの株式であり、10μlのアリコートで-20℃で保存する必要があります。 </li><li> siRNAのストックから10μLのアリコートを取り、10μMのsiRNA作業溶液を得るために、RNaseフリー水40μlを添加します。この希釈は、使用するまで-20℃で保存する必要があります。 注：siRNAの作業溶液を3回以上解凍/凍結受けるべきではありません。 </li></ol>ヒト上皮細胞株の2播種<ol><li> 37℃、5％CO 2の加湿インキュベーター中で組織培養プレート中の細胞単層として増殖するヒト上皮細胞株から増殖培地を取り除き。 </li><li> RT PBS（100ミリメートルプレートのための10mlのPBS）で1回プレートに細胞を洗浄します。 </li><li>プレートから細胞を剥離するためには、0.25％トリプシン/ 0.53 mMのEDTA溶液2mlを添加し、細胞型に応じて3~5分間インキュベートします。 </li><li>剥離した細胞を回収する前に、10％ウシ胎児血清を含む完全増殖培地2mlを添加することによりトリプシンを中和します。 </li><li> 15mlチューブに細胞を移します。 </li><li>スイングバケットローターで4℃で3分間、320〜330×gで細胞を遠心します。 </li><li>抗生物質を含まない培地5ml中に細胞ペレットを再懸濁します。 </li><li> Burkerチャンバー中または製造者の指示に従って、自動計数装置を用いて顕微鏡下で細胞を数えます。 </li><li> 6ウェルプレート中で細胞を24時間（0.5〜1×10ウェルそれぞれについて、培地2mlで6個の細胞）の後に約80％コンフルエントになるように種子。播種する細胞の最適数は、特定の細胞株および成長速度に応じて変化し得ます。 注：抗生物質が負transfeの効率を妨害するので、この時点では、培地中の抗生物質を追加しないでくださいction。これは、BRCA2のsiRNAトランスフェクションの高い効率を達成するために、低培養継代（&lt;10）の細胞であることが非常に重要です。 </li></ol> siRNAで3トランスフェクト細胞<ol><li>二十四時間細胞をプレーティングした後、トランスフェクションの最初のラウンドに進みます。 <ol><li>低血清培地130μlの脂質ベースの10特異的なsiRNAトランスフェクション試薬の6μLを希釈します。 </li><li>低血清培地130μlの10μMのsiRNA（30ピコモル）の3μLを希釈します。 </li><li>希釈されたトランスフェクション試薬で希釈したsiRNAを組み合わせて、室温で5〜10分間インキュベートします。 </li><li>インキュベーションの間、細胞から培地を除去し、37°C​​（抗生物質を含まない）に予め温めておいた新鮮な培地2mlを加えます。 </li><li> （この量は6ウェルプレートの1ウェルのためのものである）を細胞にsiRNAをトランスフェクション試薬複合体の250μlを添加します。 </li><li> 37の加湿インキュベーター中で細胞をインキュベート76; C、5％CO 2。 </li></ol></li><li> 24時間後、ステップ3.1.2には、次の変更で3.1.1-3.1.5の手順を繰り返すことにより、トランスフェクションの第二ラウンドを続行：低血清培地130μlの10μMのsiRNA（50ピコモル）の5μLを希釈します。 <ol><li>分析の前に、37℃で1〜2日間細胞をインキュベートします。 注：トランスフェクションし、第二ラウンドで高いのsiRNA /トランスフェクション試薬比の使用の2ラウンドは、BRCA2サイレンシングの高効率を得るために不可欠です。 </li></ol></li></ol>イムノブロット分析のための4を溶解細胞<ol><li>非イオン性、非変性洗剤オクチルフェノキシポリエトキシエタノール、5mMのピロリン酸ナトリウム、1mMのオルトバナジン酸ナトリウム、10mMのフッ化ナトリウム、2mMのフェニルメチルスルホニルフルオリド、を10μg/ mlのロイペプチン、10を1％含有PBS新鮮な溶解緩衝液（pH 7.4）を準備/ mlのアプロチニン。氷の上に保管してください。 </li><li>細胞およびWからメディアを削除しますプレート内の冷PBS（2ミリリットル/ウェル）で1回、それらを灰。任意のPBSのキャリーオーバーを防止するために、完全にプレートから取り外します。 </li><li>各ウェルに200μlの溶解緩衝液を追加します。 注：細胞は、他のアッセイにも使用する必要がある場合、ステップ2.1〜2.6に記載し、使用する用途に応じて適切な緩衝液/培地中で細胞を再懸濁したように、細胞をトリプシン処理。 </li><li>静かに均一に溶解バッファーを配布し、回転子に4℃で15分間インキュベートする板を回転させます。 </li><li>セルスクレーパーを用いて氷上でマイクロ遠心チューブ内の細胞溶解物を収集します。 </li><li> 4℃で25分間、17,900×gで遠心分離し、新しいチューブに上清を収集します。 </li><li>製造業者の指示に従って市販のタンパク質アッセイを用いてタンパク質濃度を測定します。 </li></ol> 5. BRCA2のイムノブロット分析<ol><li> Bを実行して20倍のトリス - 酢酸SDSの50ミリリットルを希釈することにより、ランニング緩衝液を調製します蒸留水950ミリリットルでuffer（50mMのトリシン、50mMのトリス塩基、0.1％SDS、pHは8.24）。 </li><li>次のように試料を調製：20の最終容量まで15全細胞溶解物のμgの、4×サンプルバッファー5μlのpHは8.4でドデシル硫酸リチウムを含む、10倍のサンプル還元剤（0.5 M DTT）を2μl、および溶解バッファーを追加μlの。 注：（5μgのまで）全タンパク質のより少量を使用した場合、このプロトコルは、正常細胞株でBRCA2の検出を可能にします。 </li><li> 10分間55℃でサンプルを変性。 注：これは、55℃を使用することが重要です。 BRCA2タンパク質は感熱11であるため、より高い変性温度は、無傷の完全長（390キロダルトン）BRCA2タンパク質の一貫した損失をもたらします。 </li><li>製造者の指示に従って電気泳動チャンバーを設定します。 </li><li>サンプルおよびプレキャストゲル（トリス - 酢酸3から8パーセント）の高分子量プレ染色タンパク質標準の10μLをロードし、AB、120 Vで動作します2時間12アウト。 55kDaの青いマーカーがプレキャストゲルを離れる前に、電気泳動実行を停止します。 </li><li> 20X転送バッファの50ミリリットル、100％メタノール850 mlの水100mlで転送バッファを準備します。 </li><li>蒸留水で洗浄し、少なくとも5分間、転写緩衝液中で平衡化し、5分間、100％メタノール中に浸漬することによりPVDF膜（0.45μmの孔径）をアクティブにします。使用するまで4℃で転送バッファ内の転送装置のスポンジを浸します。 </li><li>電気泳動の実行が終了すると、次の順序（ボトムアップ）での転送スタックのサンドイッチ組み立てる：転送バッファに飽和3スポンジを、1 3MMグレード紙は、転送バッファ、ゲル、活性化されたPVDF膜、1 3mmの飽和しますトランスファー緩衝液に飽和等級紙、転送バッファ13,14飽和3スポンジ。装置内にスタックを配置し、4℃で一晩4℃で180 mAで4時間、350 mAでタンパク質を転送します。 注：ビーイングBRCA2非常に大きなタンパク質は、高分子量のタンパク質の完全な移動を確実にするために、4時間または一晩に1時間（メーカー、最も転送プロトコルによって示唆されるように）からの転送時間を延長することが重要です。また、原因長い転送時間には、過熱を最小化し、サンドイッチ分解を容易にするために、4℃の冷室でブロッティングを行うことが必須です。セミドライトランスファーシステムは、大きなタンパク質を転送するための良いものではありません。 </li><li>転写後、TBSで膜を洗浄し、0.25％（TBS-T）を30μlの抗BRCA2ウサギポリクローナル抗体と共に4℃で一晩、5％脱脂粉乳およびプローブを含むTBS-Tween中で室温で1時間、それを阻止します（ [：300（v / v）の希釈液1] 9ミリリットルTBS-T + 5％脱脂粉乳中で希釈した200μg/ ml）を、。 </li><li>その後TBS 10mlに希釈した西洋ワサビペルオキシダーゼ結合抗ウサギ二次抗体の1μlの1時間、フィルターをインキュベートし、TBS-Tを用いて膜を3回（10分毎）で洗浄-T + 5％の脱脂粉乳。その後、TBS-Tで膜を3回（各10分）を洗浄します。 </li><li>製造業者の説明書に従って、増強化学発光（ECL）ウエスタンブロッティング検出試薬を用いて、BRCA2タンパク質を明らかにし、ECLによって免疫検出を実行します。高解像度ECLフィルム上immunochemiluminescentバンドを可視化。 </li><li> 10 1,000抗チューブリン抗体（10μL：1に希釈したチューブリンをβに対するモノクローナル抗体を用いて、ローディングコントロールとして同じフィルタ上に、βチューブリン（55kDaの）のように、ハウスキーピング遺伝子に対する抗体を用いて免疫検出を行いますRTで1時間mlのTBS-T + 5％脱脂粉乳）。西洋ワサビペルオキシダーゼ結合抗マウスの代わりに抗ウサギ二次抗体を使用したことを除き、洗浄し、BRCA2（5.10から5.11）について記載したように二次抗体をインキュベートします。 </li><li> immunochemiluminescentバンドと感動フィルムのデジタル画像を取得し、専用の画像解析ソフトウェアによって、バンドの強度を分析する（ 例えば。、ImageJの）。 </li></ol>

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The authors have nothing to disclose.

女性と男性の乳癌、卵巣癌、前立腺癌、膵臓癌、及びメラノーマ3,4を含むいくつかの癌型のリスク増加にBRCA2遺伝子リードの生殖細胞変異は、多くの研究は、生物学的機能を理解するために行われているのでBRCA2タンパク質の。これらの研究のほとんどが原因BRCA2のような巨大なタンパク質の分析における技術的困難に遺伝ベースの主です。 BRCA2タンパク質の発現をサイレンシ?...

BRCA2（乳癌感受性遺伝子-2）遺伝子は、リコンビナーゼ酵素のRad51 1の機能を調節することにより、DNA二本鎖切断の修復に重要な役割を演じる腫瘍抑制タンパク質をコードします。 BRCA2はまた、転写の調節および細胞周期の制御2に関係しています。 BRCA2遺伝子に生殖細胞変異は、男性に女性と前立腺癌における乳癌および卵巣癌に常染色体優性の感受性を誘導するだけでなく、他の癌タイプ3,4の素因。しかし、抑制またはDNA損傷5-7を引き起こす化学療法剤に対する癌細胞の脆弱レンダリングしてもよいBRCA2の機能を低下させる癌、BRCA2変異を開発するリスクの増加にもかかわらず。 BRCA2タンパク質レベルの低下は、そのBRCA2タンパク質を示唆し、いくつかの癌の種類で検出されているものの、散発性の癌はBRCA2変異 （&lt;3％）の低速度を示します非突然変異依存機構7,8を介して散発性の癌において腫瘍形成の間に失われることがあります。したがって、完全に癌生物学の文脈において、ならびに他の生物学的環境においてBRCA2の機能を理解することが重要です。 哺乳動物細胞における遺伝子の新規な機能を識別するために使用される強力なツールは、その発現を抑制することです。一例として、正常上皮細胞の様々なBRCA2の発現を抑制することは、最近アノイキス耐性の調節、癌細胞浸潤性および転移能9の取得中に重要なステップとして、BRCA2の新規機能の同定につながりました。遺伝子サイレンシングは、細胞小干渉（SI）RNAまたは特定の遺伝子を標的とする短いヘアピン（SH）RNA分子のいずれかで導入することによって達成することができます。高いsiRNAの効力とその使いやすさは重要な生物学的プロセスに新たな洞察を得ることを目的とした実験をサイレンシングするため、それを優先ツールを作ります新たな治療標的を同定するためにND。しかし、遺伝子発現の効率的なノックダウンは、すべての遺伝子のために容易に達成できないことがあり、細胞の種類に応じて非常に変化してもよいです。細胞内タンパク質のレベルの減少は、調査中の最も関連性の表現型であるため、遺伝子産物の解析をイムノブロッティングにより遺伝子サイレンシングを定量化することが重要です。これに関しては、BRCA2タンパク質は、さらに課題を提示：大きなタンパク質（約390キロダルトン）をされ、技術的な困難が免疫ブロット法を含む、従来の生化学的解析のために存在します。 ここでは、ヒト上皮細胞株におけるBRCA2の効率的なサイレンシングのためと分析をイムノブロッティングによりノックダウンBRCA2タンパク質の迅速かつ成功した検出のために最適化されたプロトコルを報告しています。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>BRCA2 siRNA</td>
			<td>Dharmacon</td>
			<td>L-003462-00</td>
			<td>SMARTpool: ON-TARGETplus BRCA2 siRNA</td>
		</tr>
		<tr>
			<td>Ctrl siRNA</td>
			<td>Dharmacon</td>
			<td>&#160;D-001810-10-05</td>
			<td>ON-TARGETplus Non-targeting Pool</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Reagent&#160;</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/13778075">13778075</a></td>
			<td>Transfection Reagent</td>
		</tr>
		<tr>
			<td>OPTI-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td>Medium for transfection procedure</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate 3-8% 1,5 mm Gel precast</td>
			<td>Life Technologies</td>
			<td>EA0378</td>
			<td>Gel for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample buffer&#160;</td>
			<td>Life Technologies</td>
			<td>NP0007</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Sample Reducing agent (10x)</td>
			<td>Life Technologies</td>
			<td>NP0004</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Antioxidant</td>
			<td>Life Technologies</td>
			<td>NP0005</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>HiMark Pre-stained protein standard</td>
			<td>Life Technologies</td>
			<td>LC5699</td>
			<td>Prestained marker for gel electrophoresis</td>
		</tr>
		<tr>
			<td>XCell SureLoc Mini-Cell&#160; System</td>
			<td>Life Technologies</td>
			<td>EI0002</td>
			<td>Equipment for protein electrophoresis/transfer</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate SDS Running Buffer</td>
			<td>Life Technologies</td>
			<td>LA0041</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer</td>
			<td>Life Technologies</td>
			<td>NP0006-1</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>BRCA2 H-300 rabbit polyclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8326</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Beta-tubulin monoclonal antibody</td>
			<td>Sigma Aldrich</td>
			<td>T4026-100UL</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Skim Milk powder</td>
			<td>Sigma Aldrich</td>
			<td>70166</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico/Femto Chemiluminescent Substrate</td>
			<td>Pierce Thermo Scientific</td>
			<td>34080/34096</td>
			<td>Chemiluminescence system</td>
		</tr>
		<tr>
			<td>PNT1A cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>95012614</td>
			<td>PNT1A human, normal prostate epithelium immortalized with SV40</td>
		</tr>
		<tr>
			<td>Nthy cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>90011609</td>
			<td>Nthy-ori 3-1 Cell Line human, thyroid follicular epithelial cells</td>
		</tr>
		<tr>
			<td>Cell Proliferation Kit I (MTT)</td>
			<td>Roche</td>
			<td>11465007001</td>
			<td>Kit for cell proliferation assays</td>
		</tr>
	</tbody>
</table>

silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells

従来の生化学的分析による腫瘍抑制タンパク質BRCA2とその検出のサイレンシングは、ヒトのBRCA2タンパク質（約390キロダルトン）のサイズが大きいために大きな技術的課題を表します。我々は、ヒト上皮細胞株において、それぞれ、ヒトのBRCA2を沈黙し、内因性BRCA2タンパク質を検出するための標準的なsiRNAトランスフェクションおよびイムノブロッティングのプロトコルの変更を報告しています。重要なステップは、トランスフェクション試薬の比が高いsiRNAを、同じ実験内のsiRNAトランスフェクションの2その後のラウンドがあります。抗BRCA2抗体を用いたイムノブロット分析によって判断されるように、我々は一貫して人間のBRCA2遺伝子の70％以上のサイレンシングを達成するための標準プロトコルへのこれらおよびその他の変更を使用しました。また、代わりに、従来の70〜100°Cおよび免疫ブロット法の他の技術的な最適化の55℃での細胞溶解物の変性は、無傷のBRCA2タンパク質の検出が可能であってもWH出発物質が非常に少量エン入手可能であるか、またはBRCA2タンパク質の発現レベルが非常に低い場合。ヒト細胞におけるBRCA2の効率的なサイレンシングは、腫瘍中の病原性BRCA2変異によって影響を受ける可能性がある新たな分子経路および細胞機能を調べるために、腫瘍細胞を含む、無傷のBRCA2と細胞内でBRCA2の機能を破壊するための貴重な戦略を提供しています。効率的なサイレンシングおよびBRCA2のような他の「大規模な」タンパク質の分析のためにこのプロトコルの適応が容易に達成する必要があります。

細胞の機能にサイレンシングBRCA2の影響を調べるために、生化学的/生物学的ア ​​ッセイに進む前に、最初のステップは、BRCA2のsiRNA（ 図1Aと並んでスクランブルsiRNA（非標的とするsiRNA）側を使用してサイレンシングの特異性を確認することです）。これは、BRCA2サイレンシングの高い効率を得るために高いのsiRNA /トランスフェクション比でsiRNAトランスフェ...

Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

のサイレンシング<em&gt; BRCA2</em&gt;ヒト培養細胞における新規BRCA2-調節される生物学的機能を同定するために、

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

silencing-brca2-to-identify-novel-brca2-regulated-biological

Research

JoVE Journal

Biology

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

9.0K ビュー.  National Research Council. Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. 

ビデオ: Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

初代培養海馬ニューロンにおける高密度コア小胞のライブイメージング

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

生物学

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

遺伝子発現のDNA相互作用、ディスカバー転写レギュレータ、および新規抗腫瘍剤を同定：タンパク質を研究するために、定量的アッセイ

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

培養細胞における小胞体サブドメインの可視化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕獲化合物質量分析 - 小説のc-ジ-GMPエフェクタータンパク質を同定するための強力なツール

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

培養哺乳動物細胞内の生物学的経路のユビキチンおよびユビキチン様システムレギュレータを識別するために、siRNAのスクリーニング

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

インスリン刺激に対するグルコース取り込み測定と応答<em&gt;インビトロ</em&gt;培養ヒト初代筋管

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...